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Title: Single molecule studies of F1-ATPase and the application of external torque
Author: Bilyard, Thomas
ISNI:       0000 0004 2675 9429
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2009
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F1-ATPase, the sector of ATP synthase where the synthesis of cellular ATP occurs, is a rotary molecular motor in its own right. Driven by ATP hydrolysis, direct observation of the rotation of the central axis within single molecules of F1 is possible. Operating at close to 100% efficiency, F1 from thermophilic Bacillus has been shown to produce ~40pN˙nm of torque during rotation. This thesis details the groundwork required for the direct measurement of the torque produced by F1 using a rotary angle clamp, an optical trapping system specifically designed for application to rotary molecular motors. Proof-of-concept experiments will be presented thereby demonstrating the ability to directly manipulate single F1 molecules from Escherichia coli and yeast mitochondria (Saccharomyces cerevisiae), along with activation of F1 out of its inhibited state by the application of external torque. Despite in-depth knowledge of the rotary mechanism of F1 from thermophilic Bacillus, the rotation of F1 from Escherichia coli is relatively poorly understood. A detailed mechanical characterization of E.coli F1 will be presented here, with particular attention to the ground states within the catalytic cycle, notably the ATP-binding state, the catalytic state and the inhibited state. The fundamental mechanism of E.coli F1 appears to depart little from that of F1 from thermophilic Bacillus, although, at room temperature, chemical processes occur faster within the E.coli enzyme, in line with considerations regarding the physiological conditions of the different species. Also presented here is the verification of the rotary nature of yeast mitochondrial F1. The torque produced by F1 from thermophilic Bacillus, E.coli and yeast mitochondria is the same, within experimental error, despite their diverse evolutionary and environmental origins.
Supervisor: Berry, Richard M. Sponsor: Engineering and Physical Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Physical Sciences ; Biophysics ; Biophysical chemistry ; Enzymes ; Laser Spectroscopy ; Membrane proteins ; Microscopy ; Protein chemistry ; Surface chemistry ; Physics ; Condensed Matter Physics ; Stochastic processes ; Probability ; ATP synthase ; single ; molecule ; study ; assay ; rotation ; rotary ; molecular ; motor ; optical ; trap ; tweezer ; laser ; back ; focal ; plane ; interferometry